An astrocytic signaling loop for frequency-dependent control of dendritic integration and spatial learning

Dendrites of hippocampal CA1 pyramidal cells amplify clustered glutamatergic input by activation of voltage-gated sodium channels and N-methyl-D-aspartate receptors (NMDARs). NMDAR activity depends on the presence of NMDAR co-agonists such as D-serine, but how co-agonists influence dendritic integration is not well understood. Using combinations of whole-cell patch clamp, iontophoretic glutamate application, two-photon excitation fluorescence microscopy and glutamate uncaging in acute rat and mouse brain slices we found that exogenous D-serine reduced the threshold of dendritic spikes and increased their amplitude. Triggering an astrocytic mechanism controlling endogenous D-serine supply via endocannabinoid receptors (CBRs) also increased dendritic spiking. Unexpectedly, this pathway was activated by pyramidal cell activity primarily in the theta range, which required HCN channels and astrocytic CB1Rs. Therefore, astrocytes close a positive and frequency-dependent feedback loop between pyramidal cell activity and their integration of dendritic input. Its disruption in mice led to an impairment of spatial memory, which demonstrated its behavioral relevance.

representative filtered traces of the ratio of GCaMP5g and tdTomato (background-subtracted and normalized to baseline) are illustrated for a nonresponding (asterisk, grey trace) and a responding neuron (arrow, blue trace). Cells were considered responders if the ratio increased more than 25 % over baseline (dashed line). j) The reliability of evoking single unit activity did not depend on the alveus stimulation frequency. When a pyramidal cell single unit response was identified blindly during 10 Hz stimulation of the alveus, several trains of stimuli were delivered at three frequencies (4, 10 and 40 Hz) and the probability of observing a unit spike was calculated. No significant differences were detected (4 Hz: 97.3 ± 1.9 %, 10 Hz: 99.5 ± 0.3 %, 40 Hz: 97.9 ± 1.4, n = 16 cells from 7 independent experiments, F(1.01,15.13) = 1.55, p = 0.23 one-way repeatedmeasures ANOVA).
Data are expressed and displayed as mean ± s.e.m. or in box plots. In the latter, the box indicates the 25th and 75th, the whiskers the 5th and 95th percentiles, the horizontal line in the box the median and the mean is represented by a filled circle. Source data are provided as a Source Data file. a) Threshold stimulus during baseline, 20 Hz alveus stimulation, and five minutes later (baseline: 0.38 ± 0.03 µA, 20 Hz: 0.34 ± 0.03 µA, 5 min: 0.41 ± 0.04 µA, n = 9, χ 2 (2) = 9.06, p = 0.011 Friedman test; post-hoc two-sided Wilcoxon signed-rank tests: z = 2.12, p = 0.031 for baseline vs. 20 Hz, z = 1.26, p = 0.21 for baseline vs. 5 min, z = 2.61, p = 0.0039 for 20 Hz vs. 5 min). b) Analysis of the slow component of dendritic spikes of the same recordings. n = 8 because somatic spikes prevented analysis of the slow component in one recording (baseline: 9.75 ± 1.37 mV, 20 Hz: 11.08 ± 1.81 mV, 5 min: 9.41 ± 1.56, n = 8, F(2,14) = 5.80, p = 0.015 oneway repeated measures ANOVA; post-hoc Fisher's LSD test, t(14) = 2.57, p = 0.022 for baseline vs. 20 Hz, t(14) = 0.66, p = 0.52 for baseline vs. 5 min, t(14) = 3.22, p = 0.0061 for 20 Hz vs. 5 min). c) Sample traces for baseline (black), 20 Hz alveus stimulation (yellow) and 5 min after (grey). The upper panel (black border) shows traces for all three conditions recorded with the stimulus intensity that just evoked a dendritic spike under baseline conditions (threshold stimulus). Note that during alveus stimulation the slow component is increased. The lower panel (yellow border) shows traces for all three conditions recorded with the stimulus intensity that just evoked a dendritic spike during 20 Hz alveus stimulation. Because alveus stimulation reduces the threshold stimulus, no dendritic spikes occur at that simulation intensity under baseline conditions and 5 minutes after alveus stimulation. Insets display dV/dt with the same color coding. Scale bars 2 mV and 20 ms and in insets 2 mV/ms and 5 ms.
Data are expressed and displayed as mean ± s.e.m. Source data are provided as a Source Data file.   Data are expressed and displayed as mean ± s.e.m. or in box plots. In the latter, the box indicates the 25th and 75th, the whiskers the 5th and 95th percentiles, the horizontal line in the box the median and the mean is represented by a filled circle. Source data are provided as a Source Data file.
The Kolmogorov-Smirnov test was chosen for c and d because data sets do not follow a normal distribution preventing further analysis with, for example, a two-way repeated measure ANOVA.
Data are expressed as mean ± s.e.m. in the legend and displayed in box plots. The box indicates the 25th and 75th, the whiskers the 5th and 95th percentiles, the horizontal line in the box the median and the mean is represented by a filled circle. Source data are provided as a Source Data file. b) The paired-pulse ratio of fEPSP was recorded at several inter-stimulus intervals. No statistically significant differences were found between groups (WT: n = 7 from 4 animals, sham: n = 12 from 5 animals; aCB1KO: n = 14 from 6 animals; two-way repeated measures ANOVA, between groups, F(2,30) = 0.77, p = 0.39) c) Population spikes were recorded from the CA1 pyramidal layer and evoked by stimulation of CA3-CA1 synaptic connections at various stimulation intensities. No statistically significant differences between groups (WT: n = 46 from 20 animals, sham: n = 32 from 17 animals; aCB1KO: n = 30 from 14 animals; two-way repeated measures ANOVA, between groups, F(2,105) = 1.59, p = 0.21).
Data are expressed and displayed as mean ± s.e.m. Source data are provided as a Source Data file.
i) The total distance travelled by mice declined rapidly during place avoidance learning but did not differ between experimental groups (two-way repeated measures ANOVA; for days F(4,120) = 186.20, p < 0.001, post-hoc Tukey test p < 0.001 as indicated by asterisks; for groups F(1,30) = 0.32, p = 0.58; for interaction F(4,120) = 1.23, p = 0.30; n = 17 and 15 for sham and aCB1KO, respectively). j) Sample tracks at day 4. The new air puff location is marked by arrows. Note that the shamtreated animals successfully and rapidly avoid the air puff. k) Number of air puff activations in both groups on day 4. In contrast to Fig. 9, animals that did not fully explore the arena on day 3 (no air puff in any location) were excluded from this analysis, because they had no opportunity to notice the absence of an air puff on day 3. This did not change the results qualitatively. Sham-treated animals activated fewer air puffs, i.e., avoided the new air puff area more successfully (sham: 3.75 ± 0.57, n = 12; aCB1KO: 6.14 ± 0.62, n = 14; t(24) = 2.82, p = 0.0096, two-sided Student's t-test). l) How quickly animals avoided the new air puff location was quantified by measuring the time between the first triggered air puff and the last, after which the air puff area was completely avoided. This time to avoidance is low when mice rapidly acquire the new location of the aversive stimulus. Sham-treated animals displayed a significantly shorter time to avoidance (sham: 259.6 ± 38.2 s, n = 17; aCB1KO: 389.2 ± 32.3 s, n = 15; t(30) = 2.55, p = 0.016, two-sided Student's t-test).
Data are expressed and displayed as mean ± s.e.m. or in box plots. In the latter, the box indicates the 25th and 75th, the whiskers the 5th and 95th percentiles, the horizontal line in the box the median and the mean is represented by a filled circle. Source data are provided as a Source Data file.